Compositions and methods for treating oculopharyngeal muscular dystrophy

ABSTRACT

The present invention provides intravenous compositions of trehalose for the treatment of signs and symptoms of oculopharyngeal muscular dystrophy (OPMD).

RELATED APPLICATIONS

This application is a continuation in part of PCT/IL2014/050411, filedMay 7, 2014, which claims priority to and benefit of U.S. ProvisionalApplication No. 61/820,278, filed May 7, 2013, and this applicationclaims priority to and benefit of U.S. Provisional Application No.61/990,027, filed May 7, 2014, the contents of which are each hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates generally intravenous compositions of trehalosefor the treatment of signs and symptoms of aggregation disease ordisorders including oculopharyngeal muscular dystrophy (OPMD).

BACKGROUND OF THE INVENTION

Oculopharyngeal Muscular Dystrophy (OPMD) is a rare inherited myopathycharacterized by ptosis, severe dysphagia and proximal limb weakness.Its estimated prevalence is 1:100,000 and the largest clusters reportedwere in families of French-Canadians origin in Canada and in the US(prevalence 1:1000), Bukhara Jews in Israel (prevalence 1:600) andHispanics in New Mexico, Arizona Colorado and California [1-4]. OPMD isinherited, in most cases, as an autosomal dominant trait with completepenetrance. The disease is equally prevalent among both genders. Thegene associated with the disease has been identified. This mutationresults in production of an abnormal poly (A) binding protein nuclear 1protein (PABPN 1), a nuclear protein involved in pre-mRNApolyadenylation, transcription regulation, and mRNA nucleocytoplasmictransport.

The disease is most often diagnosed in the fifth-sixth decades of lifeand progresses throughout the patient's life. By age 70 the majority ofpatients suffer from all or some of the following symptoms: severedysphagia, ptosis, tongue atrophy and weakness, lower and upper limbproximal weakness, dysphonia, limitation in upward gaze and facialmuscle weakness. As ptosis becomes more pronounced patients adapt the“astronomer posture,” tilting of the head and upward gaze—furtheraggravating the dysphagia. The dysphagia starts with difficulty inswallowing solid food and progresses to liquids as well. As thedysphagia becomes more severe, patients become malnourished, cachectic,dehydrated and suffer from repeated aspiration pneumonia. OPMD does notseem to shorten life expectancy but is associated with severedebilitation and reduced quality of life.

There is no medical treatment or potential cure for OPMD. Currenttherapeutic strategies are confined to surgical interventions aimed atalleviating ptosis. Repeated cricopharyngeal dilatations are frequentlyused to relieve dysphagia. Myotomy of the upper esophageal sphinctermuscles has also been employed. These procedures may provide onlytemporary relief and do not affect the progression of the disease thateventually leads to severe difficulty in swallowing, recurrentaspiration with increasing risk of aspiration pneumonia and severeweight loss which are the most common causes of mortality in OPMDpatients.

Accordingly, there is an urgent need for compositions and therapeuticmethods for alleviating the signs and symptoms of oculopharyngealmuscular dystrophy.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that rapid intravenousinjection of trehalose results in cellular uptake and retention oftrehalose. Trehalose has been shown to prevent pathological aggregationof proteins within cells. Thus, the present invention provides anaqueous formulation for intravenous injection, comprising a singleactive ingredient consisting of trehalose and methods of use thereof.

In one embodiment, the trehalose formulation of the present inventionhas a pH about 4.5 to 7.0. In another embodiment, the formulationcontains less than 0.75 endotoxin units per mL. In another embodiment,the formulation contains 10% (w/v) trehalose. In another embodiment, theformulation has an osmolality of about 280-330 mOsm/kg.

The trehalose formulation of the present invention is retained in thecell for an extended period of time after intravenous administration ofthe formulation to the cell. In one embodiment, the formulation isretained in the cell for about 48-72 hours after intravenous injection.In another embodiment, the intravenous injection is completed within 120minutes.

The trehalose formulation of the present invention prevents aggregationof proteins within the cells. In one embodiment, the formulation of thepresent invention prevents aggregation of a protein that is involved inthe pathogenesis of or associated with a sign or symptom ofoculopharyngeal muscular dystrophy. In a further embodiment, theformulation of the present invention prevents aggregation of theabnormal protein PABPN1.

The present invention also provides a method of alleviating a sign orsymptom of a disease, by intravenously administering to a subject inneed thereof a formulation of the present invention. In one embodiment,the intravenous injection of the formulation is completed within 120minutes. In another embodiment, the formulation is administered onceweekly. In another embodiment, the formulation is administered at 0.5gram trehalose per kilogram body weight per day. In another embodiment,the formulation is administered between 5 to 35 grams trehalose per day.In another embodiment, the disease is oculopharyngeal musculardystrophy. In a further embodiment, the sign or symptom includes, but isnot limited to, muscle weakness, formation of protein aggregates (e.g.,PABPN1 aggregates), and formation of pathological skeletal musclefibers.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Mean Plasma Concentrations (ng/mL) of Trehalose Following SingleIntravenous or Oral Administration of Trehalose Dihydrate at 1000 mg/kgto Male SD Rats

FIG. 2 Mean Plasma and Muscle Concentrations (ng/mL or ng/g) ofTrehalose Following Single Intravenous Administration of TrehaloseDihydrate at 1000 mg/kg to Male SD Rats

FIG. 3 Mean Plasma and Muscle Concentrations (ng/mL or ng/g) ofTrehalose Following Single Oral Administration of Trehalose Dihydrate at1000 mg/kg to Male SD Rats

FIG. 4 Plasma Glucose Ratios of Drug Treated Group to That of VehicleGroup Following Intravenous or Oral Administration of Vehicle orTrehalose Dihydrate at 1000 mg/kg to Male SD Rats

FIG. 5 Mean Plasma Glucose Levels (mmol/L) Following Intravenous or OralAdministration of Vehicle or Trehalose Dihydrate at 1000 mg/kg to MaleSD Rats

FIG. 6 Individual Plasma Glucose Levels (mmol/L) Following IntravenousAdministration of Vehicle to Male SD Rats

FIG. 7 Individual Plasma Glucose Levels (mmol/L) Following OralAdministration of Vehicle to Male SD Rats FIG. 9 Mean Plasma. GlucoseLevels (mmol/L) Following Oral Administration of Trehalose Dihydrate at1000 mg/kg or Vehicle to Male SD Rats

FIG. 8 Mean Plasma Glucose Levels (mmol/L) Following IntravenousAdministration of Trehalose Dihydrate at 1000 mg/kg or Vehicle to MaleSD Rats

FIG. 9 Mean Plasma Glucose Levels (mmol/L) Following Oral Administrationof Trehalose Dihydrate at 1000 mg/kg or Vehicle to Male SD Rats

DETAILED DESCRIPTION

The present invention provides aqueous solutions of trehalose forintravenous injection. Surprisingly, rapid intravenous administration oftrehalose results in cellular uptake and retention 48-72 hours.Accordingly, the invention provides methods for treating the signs andsymptoms of oculopharyngeal muscular dystrophy (OPMD) by intravenousadministration or trehalose. In other aspects, the compositions andmethods disclosed herein may be used to treat other aggregation diseaseor disorders such as poly-alanine aggregation disorder, poly-glutamineaggregation disorder and a tauopathy. In particular aspects, the diseaseor disorder is spinocerebellar ataxias (SCA), Friedreich's ataxia,spinal and bulbar muscular atrophy (SBMA), Huntington's disease,Parkinson's disease, Alzheimer's disease and amyotrophic lateralsclerosis (ALS), dentatorubral-pailidoluysian atrophy (DRPLA), Pick'sdisease, Corticobasal degeneration (CBD), Progressive supranuclear palsy(PSP) and Frontotemporal dementia and parkinsonism linked to chromosome17 (FTDP-17).

Before describing the present invention in detail, it is to beunderstood that unless otherwise indicated, this invention is notlimited to particular dosages, formulations or methods of use, as suchmay vary. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to be limiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example, “adosage form” refers not only to a single dosage form but also to acombination of two or more different dosage forms, “an active agent”refers to a combination of active agents as well as to a single activeagent, and the like.

As used in the specification and the appended claims, the terms “forexample,” “for instance,” “such as,” “including” and the like are meantto introduce examples that further clarify more general subject matter.Unless otherwise specified, these examples are provided only as an aidfor understanding the invention, and are not meant to be limiting in anyfashion.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which the invention pertains. Although any methods and materialssimilar or equivalent to those described herein may be useful in thepractice or testing of the present invention, preferred methods andmaterials are described below. Specific terminology of particularimportance to the description of the present invention is defined below.

When referring to a compound of the invention, applicants intend theterm “compound” to encompass not only the specified molecular entity butalso its pharmaceutically acceptable, pharmacologically active analogs,including, but not limited to, salts, polymorphs, esters, amides,prodrugs, adducts, conjugates, active metabolites, and the like. Whenthe term “compound” is used, then, it is to be understood thatapplicants intend to include that compound per se as well aspharmaceutically acceptable, pharmacologically active salts, polymorphs,esters, amides, prodrugs, adducts, conjugates, metabolites, and othersuch derivatives, analogs and related compounds.

The terms “treating” and “treatment” as used herein refer to reductionin severity and/or frequency of signs or symptoms, elimination of signsor symptoms and/or underlying cause, prevention of the occurrence ofsymptoms and/or their underlying cause (e.g., prophylactic therapy), andimprovement or remediation of damage.

By the terms “effective amount” and “therapeutically effective amount”of a compound of the invention is meant a nontoxic but sufficient amountof the drug or agent to provide the desired effect.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating OPMD. Alleviation of one or more signsor symptoms of OPMD indicates that the compound confers a clinicalbenefit.

The term “injection” as used herein refers to a bolus injection, slowbolus injection over several minutes, or prolonged infusion, or severalconsecutive injections/infusions that are given at spaced apartintervals. Generally, since rate of administration of the disclosedinjectable formulation is also determined in consideration of the levelof endotoxins in the formulation, a per administration “injection” of aneffective dose of the trehalose can be divided into spaced apartinjections of lower amounts of formulation, until the whole of theeffective dose is administered. Such spaced apart injections per asingle administration are also referred to herein as “peradministration” or “per administration injection” or the like, or inother words, a single administration can include several injections orprolonged infusion. The administration of the aqueous injectablesolution of trehalose, particularly for the treatment of OPMD, asdisclosed herein is completed in no more than 120 minutes, and the rateof administration is such that the maximum endotoxin level is no morethan 5 EU per kilogram of body weight of the patient per hour.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. When the term “pharmaceutically acceptable” isused to refer to a pharmaceutical carrier or excipient, it is impliedthat the carrier or excipient has met the required standards oftoxicological and manufacturing testing or that it is included on theInactive ingredient Guide prepared by the U.S. Food and Drugadministration.

By “patient” is meant any animal for which treatment is desirable.Patients may be mammals, and typically, as used herein, a patient is ahuman individual.

The term “about” as used herein indicates values that may deviate up to1%, more specifically 5%, more specifically 10%, more specifically 15%,and in some cases up to 20% higher or lower than the value referred to,the deviation range including integer values, and, if applicable,non-integer values as well, constituting a continuous range.

Compositions

“Trehalose” is a stable, nonreducing disaccharide with two glucosemolecules linked in a 1,1 configuration.

Trehalose is well known for its protein-stabilizing properties [10,11].It is used extensively in many applications as a stabilizer of frozenfood, in freeze-drying of biological systems and cells, as a stabilizerof therapeutic parenteral proteins and as an excipient in tablets and IVsolutions. Trehalose is recognized as a GRAS (Generally Regarded asSafe) food ingredient by the FDA and is listed on the USP-NF (UnitedStates Pharmacopoeia National Formulary), EP (European Pharmacopoeia)and JP (Japanese Pharmacopoeia). This disaccharide chemical chaperonehas been shown to prevent pathological aggregation of proteins withincells in several diseases associated with abnormal cellular-proteinaggregation such as Huntington's disease, spinocerebellar ataxia,Parkinson and Alzheimer disease.

OPMD is characterized by intracellular aggregation of the abnormalprotein PABPN1 by products of which are considered by most authoritiestoxic [12]. Trehalose was found effective in reducing the aggregationand toxicity of mutant PABPN1 proteins in OPMD cell models. Furthermore,treatment of an OPMD mouse model with trehalose resulted in theattenuation of muscle weakness, decreased aggregate formation and areduced number of pathological skeletal muscle fibers [13]. As such, itwas hypothesized that trehalose might be useful for treatment of OPMD.

Like all disaccharides, trehalose is metabolized at the epithelial brushborder to two D-glucose molecules. Less than 0.5% of ingested trehaloseis absorbed into the blood stream where it is further metabolized byliver and kidney by trehalase. Oral trehalose in amounts exceeding 40-50gram per day causes diarrhea and bloating. Thus in order to achievetherapeutic amounts of trehalose in the muscle cells it was necessary tocircumvent the massive metabolism in the GI tract. Therefore theinventors developed an I.V. solution of trehalose.

To date, the safety and toxicity of trehalose has been extensivelyinvestigated, and the substance was found to be safe when administeredboth orally and intravenously, in doses that are substantially higherthan the intended therapeutic dose.

Accordingly, in one embodiment, the compositions described hereincomprise trehalose as the sole active agent. Furthermore, in oneembodiment, the methods described herein comprise the intravenousadministration of trehalose to a patient in need thereof.

The compositions of the current disclosure comprise, as an active agent,trehalose in a pharmaceutically acceptable form. The active agent,trehalose, may be administered in the form of the compound per se, aswell as in the form of a salt, polymorph, ester, amide, prodrug,derivative, or the like, provided the salt, polymorph, ester, amide,prodrug or derivative is suitable pharmacologically. Salts, esters,amides, prodrugs and other derivatives of the active agents may beprepared using standard procedures known to those skilled in the art ofsynthetic organic chemistry and described, for example, by J. March,Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed.(New York: Wiley-Interscience, 1992). For any active agents that mayexist in enantiomeric forms, the active agent may be incorporated intothe present compositions either as the racemate or in enantiomericallypure form.

For compositions administered as aqueous or other solvent-based dosageforms (e.g., for parenteral administration), a variety of liquidcarriers may be used. Aqueous solutions may include salts, buffers, andthe like.

Salts are compounds that ionize in aqueous solutions and may beemployed, for example, to adjust the tonicity of the solution. If theactive agent is present in the form of a salt, additional salts may beadded to the composition in order, for example, to effect ion exchangewith the active agent. Salts suitable for use with the compositionsdescribed herein are known in the art and include, for example, lithium,sodium, potassium, calcium, and magnesium salts having appropriatecounterions that may be selected from chloride, bromide, iodide,carbonate, phosphate, nitrate, silicate, sulfate, phosphite, nitrite,sulfite, and the like.

Buffers are compounds or solutions that are employed to aid inmaintaining the concentration of an analyte within a desired range. Forexample, pharmaceutically acceptable pH buffers are used to maintain theacidity or basicity of a solution within a pharmaceutically acceptablerange. Buffers for use in the compositions disclosed herein may be anyknown or hereafter discovered buffer.

Excipients are inactive ingredients that may be employed in thecompositions described herein for a variety of reasons. A wide range ofexcipients are described in the literature (e.g., Rowe et al., Handbookof Pharmaceutical Excipients, McGraw Hill, 2006).

The amount of trehalose in the compositions disclosed herein will dependon a number of factors and will vary from subject to subject. Suchfactors include the severity of the symptoms, the patients age, weightand general condition, and the judgment of the prescribing physician.

Optimally the pH of the formulation is about 4.5 to 7.0. The osmolalityof the formulation is about 280-330 mOsm/kg.

The formulation contains less than 1.0, 0.9, 0.8, 0.75, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1 or less endotoxin units per mL.

Preferably the aqueous formulation is about 50%, 40%, 30%, 20%, 10%, 5%or less trehalose (w/v).

The formulation of claim 1, wherein the formulation has an osmolality ofabout 280-330 mOsm/kg.

In one embodiment, the purified trehalose is substantially free ofcontaminants resulted from the protein used in the enzymatic preparationprocess of the trehalose, such as organic solvents used in the process,e.g., ammonium, acetonitrile, acetamide, alcohol (e.g., methanol,ethanol, or isopropanol), TFA, ether or other contaminants. In thiscontext “substantially” free of contaminants means that the contaminantcontent of the peptide at the end of the purification process ispreferably less than 0.5%, less than 0.3%, less than 0.25%, less than0.1%, less than 0.05%, less than 0.04%, less than 0.03%, less than0.02%, less than 0.01%, less than 0.005%, less than 0.003%, or less than0.001% of the total weight of the trehalose. The content of contaminantscan be determined by conventional methods such as gas chromatography.

Preferably, the residual solvents in the purified trehalose of theinvention are less than the limits set in the ICH guidelines, e.g.,IMPURITIES: GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R5) (available athttp://www.ich.Org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3C/Step4/Q3C_R5_Step4.pdf). For example, the purified trehalose contains <5000 ppm ethanol(e.g., <140 ppm), and/or <3000 ppm methanol.

Methods of Use

The compositions and methods described herein are useful in thetreatment of the signs and symptoms of OPMD. Signs and symptoms of OPMDinclude severe dysphagia, ptosis, tongue atrophy and weakness, lower andupper limb proximal weakness, dysphonia, limitation in upward gaze andfacial muscle weakness.

In one embodiment, a method is provided for treating a patient sufferingfrom OPMD. The methods of treatment involve administering atherapeutically effective amount of a composition comprising trehaloseto the patient. Administration of trehalose may be carried out using anyof the compositions, modes of administration, and dosage forms describedherein.

Dosages

Pharmaceutical formulations suitable for use in conjunction with thepresent disclosure include compositions wherein trehalose is containedin a “therapeutically effective” amount, i.e., in an amount effective toachieve its intended purpose, such as treatment of OPMD. Determinationof a therapeutically effective amount is well within the capability ofthose skilled in the art.

Toxicity and therapeutic efficacy of the compositions described hereincan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., procedures used for determining themaximum tolerated dose (MTD), the ED₅₀, which is the effective dose toachieve 50% of maximal response, and the therapeutic index (TI), whichis the ratio of the MTD to the ED₅₀. Obviously, compositions with highTIs are the most preferred compositions herein, and preferred dosageregimens are those that maintain plasma levels of the trehalose at orabove a minimum concentration to maintain the desired therapeuticeffect. Dosage will, of course, also depend on a number of factors, thesite of intended delivery, the route of administration, frequency ofadministration, and other pertinent factors known to the prescribingphysician. The dosage range may be from each of 10, 20, 50, 75, 100,150, 200, 300 mg/Kg body weight per day up to each of 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 mg/Kg body weightper day. Generally, however, dosage will be in the range ofapproximately 0.1 grams/kg/day to 1 g/kg/day. Preferably the dose isless than 0.54 grams/kg/day.

In some embodiments the trehalose is administered such that the totaldaily dose (on a day of administration) is between about 5 grams to 50grams. In preferred embodiments the total per administration dose oftrehalose is 8, 15 or 30 grams. In particular embodiments the trehaloseis administered as a single dose of 5, 8, 15, 30, 40 or 50 grams.

In certain aspects, the dosing regimen is equal doses. In other aspects,gradually increasing doses, or gradually decreasing doses may be used.For example, in certain aspects, a subsequent dose may be greater orlesser than a prior dose by about 10%, 20%, 30%, 40%, 50%, or about100%.

Administration

Administration is accomplished such that that the maximum endotoxinlevel is less than 5 EU per kilogram of body weight per hour. Inparticular aspects, the endotoxin level is less than about 1, 2, 3, orless than about 4 endotoxin units per kilogram of body weight per hour.

Administration is daily, weekly, biweekly or monthly. Preferably, theadministration is weekly.

Administration of the compositions described herein may be carried outas part of a treatment regimen that may include multiple instances ofadministration of trehalose-containing compositions as well asadministration of other pharmaceutically active compositions. Such aregimen may be designed as a method of treatment for OPMD, and/or as amethod of long-term maintenance of the health of a patient after havingbeen treated for OPMD (e.g., preventing recurrences). The treatmentregimen may be designed as a method of treating a subject that isasymptomatic for OPMD, that is a subject that has been geneticallydiagnosed with OPMD but does not have any symptoms. Such treatmentregimen will delay the onset of OPMD symptoms in a subject. It will beappreciated that determination of appropriate treatment regimens iswithin the skill of practitioners in the art.

Administration of the compositions described herein may be carried outusing any appropriate mode of administration and dosage form. Preferablyadministration is parenteral. The term “parenteral” as used herein isintended to include, for example, subcutaneous, intravenous, andintramuscular injection. Most preferably, the administration isintravenous. Over 99.5% of the trehalose is not absorbed into the bloodstream. In addition, oral amounts of trehalose higher than 50 g a day inhumans frequently cause diarrhea, bloating and discomfort. Thus, inparticular aspects, the trehalose may be administered as an intravenousas an aqueous formulation to address poor absorption into thebloodstream and minimized undesirable metabolic events. In specificembodiments, the pH of the formulation is about 4.5 to 7.0, theosmolality of the formulation is about 280-330 mOsm/kg, the formulationcontains less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 orless endotoxin units per mL and the aqueous formulation is about 50%,40%, 30%, 20%, 10%, 5% or less trehalose (w/v).

The trehalose may be delivered over a suitable period. In someembodiments administration is complete within from about 75 to about 120minutes, specifically within less than 90 minutes.

In certain embodiments, effective serum levels of trehalose are achievedwithin from about 10 to about 20 or 30 or 40 or 50 or 60 minutesfollowing trehalose administration. In certain embodiments, effectiveserum levels of the active ingredient are achieved within from about 5to about 20 or 30 or 40 or 50 or 60 minutes following trehaloseadministration. In certain embodiments, effective serum levels of theactive ingredient are achieved within from about 20 to about 20 or 30 or40 or 50 or 60 minutes following trehalose administration. In certainembodiments, effective serum levels of the active ingredient areachieved within about 5, 10, 15, 20, 30, 40, 50 or 60 minutes followingtrehalose administration.

Delivery Systems

Administration of trehalose for medical uses requires safe and efficientdelivery systems. The present disclosure provides delivery systems (e.g.formulations for parenteral administration) for safe delivery of avariety of substances due to their special physico-chemical features.The delivery systems significantly enhance efficiency and quality oftrehalose absorption based on its unique physicochemical features, whichenables lower concentrations or amounts of active substance to bedelivered to a subject in a biologically active form. The presentdelivery systems provide for the direct access of the active substanceto the tissues and thus provide immediate or near-immediate effects oftrehalose to the subject in need thereof.

Accordingly, in certain embodiments, the present invention provides apharmaceutical delivery system for the improved administration oftrehalose or physiologically active derivative thereof, comprising asthe active ingredient said trehalose or physiologically activederivative thereof in a suitable carrier for fast restoration of reliefof symptoms of the disease of the treated subject.

In certain embodiments, the drug delivery systems may provide the activesubstance in a controlled release mode. In certain embodiments, the drugdelivery systems of the invention may further comprises at least oneadditional pharmaceutically active agent.

The presently disclosed delivery systems can generally comprise abuffering agent, an agent which adjusts the osmolality thereof, andoptionally, one or more pharmaceutically acceptable carriers, excipientsand/or additives as known in the art. Supplementary pharmaceuticallyacceptable active ingredients can also be incorporated into thecompositions. The carrier can be solvent or dispersion medium suitablefor parenterally-administrable compositions containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants.

As indicated above, the present trehalose delivery system can beadministered in controlled-, sustained- or delayed-release formulations.Any controlled or sustained release method known to those of ordinaryskill in the art may be used with the formulations and methods of thepresently disclosed subject matter such as those described in Langer1990 [16]. Such method comprises administering a sustained-releasecomposition or a coated implantable medical device so that atherapeutically effective dose of the composition of the invention iscontinuously delivered to a subject of such a method. Sustained releasemay also be achieved using a patch designed and formulated for thepurpose. Controlled or sustained-release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines). Sustained release formulaeor devices, or any topical formulations, may additionally containcompositions to stabilize the composition or permeate physiologicalbarrier such as skin or mucous membrane. Exemplary additional componentsmay include any physiologically acceptable detergent, or solvent suchas, for example, dimethylsulfoxide (DMSO).

In certain embodiments, the trehalose in the present compositions can beformulated for sustained or controlled release over a period of at least0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certainembodiments, the trehalose in the present compositions can be formulatedfor sustained or controlled release over a period of about 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certain embodiments, thetrehalose in the present compositions can be formulated for sustained orcontrolled release over a period of between about 0.5 or 1 or 2 or 3 or4 hours and about 5, 6, 7, 8, 9, 10, 11 or 12 hours. In certainembodiments, the trehalose in the present compositions can be formulatedfor sustained or controlled release over a period of between about 5 or6 or 7 or 8 hours and about 9, 10, 11 or 12 hours.

In certain embodiments, the trehalose in the present compositions can bein immediate, fast of burst release form.

In certain embodiments, the trehalose in the present compositions can beformulated to release up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5 or 100% of the total trehalosein about 0.5, 1, 2, 3, 4, 5, 6, 7 or 8 hours. In certain embodiments,the trehalose in the present compositions can be formulated to releasenot less than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 99, 99.5 or 100% of the total trehalose in about 0.5, 1,2, 3, 4, 5, 6, 7 or 8 hours.

In certain embodiments, the trehalose in the present compositions can bein a combination of sustained or slow release and immediate or fast orburst release forms. In certain embodiments, the relative proportion ofsustained or slow release trehalose to immediate or fast releasetrehalose is, e.g., 1 to 99, 5 to 95, 10 to 90, 15 to 85, 20 to 80, 25to 75, 30 to 70, 35 to 65, 40 to 60, 45 to 55, 50 to 50, 55 to 45, 60 to40, 65 to 35, 70 to 30, 75 to 25, 80 to 20, 85 to 15, 90 to 10, 95 to 5,or 99 to 1.

In certain embodiments, a polymeric material is used to sustain orcontrol release of trehalose. In certain embodiments, the type ofpolymeric material and the amount of which is used, have a stronginfluence on the rate of release of trehalose from the presentcompositions and delivery systems. Examples of polymers include bothhydrophobic and hydrophilic polymers. Examples of hydrophobic polymersinclude, but are not limited to, ethyl cellulose and other cellulosederivatives, fats such as glycerol palmito-stearate, beeswax, glycowax,castorwax, carnaubawax, glycerol monostearate or stearyl alcohol,hydrophobic polyacrylamide derivatives and hydrophobic methacrylic acidderivatives, as well as mixtures of these polymers. Hydrophilic polymersinclude, but are not limited to, hydrophilic cellulose derivatives suchas methyl cellulose, hydroxypropylmethyl cellulose,hydroxyethylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose,sodium carboxymethylcellulose and hydroxyethyl methylcellulose polyvinylalcohol, polyethylene, polypropylene, polystyrene, polyacrylamide,ethylene vinyl acetate copolymer, polyacrylate, polyurethane,polyvinylpyrrolidone, polymethylmethacrylate, polyvinyl acetate,polyhydroxyethyl methacrylate, as well as mixtures of these polymers.Furthermore, any mixture of one or more hydrophobic polymer and one ormore hydrophilic polymer could optionally be used.

The trehalose contained in the present compositions and delivery systemsmay be entrapped in liposomes, micro- and nano-particles.

In certain embodiments, a polymeric material to be used in the presentcompositions and delivery systems is microcrystalline cellulose such as“Avicel PH 101” manufactured by FMC BioPolymer's. Alternatively, apolymeric material to be used in the present compositions and deliverysystems is hydroxypropyl methylcellulose such as “Metholose” produced byShin-Etsu Chemical Co. In certain embodiments, a polymeric material tobe used in the present compositions and delivery systems is ethylcellulose such as “Ethocel™” manufactured by The Dow Chemical Company.In certain embodiments, a polymeric material to be used in the presentcompositions and delivery systems is an acrylic polymer such as“Eudragit RS™” produced by Rohm GmbH. In certain embodiments, apolymeric material to be used in the present compositions and deliverysystems is a colloidal silicone dioxide such as “Aerosil™” manufacturedby Degussa. In certain embodiments, a polymeric material to be used inthe present compositions and delivery systems is a Poly (Vinyl Acetate)such as “Kollicoat SR” manufactured by BASF. In certain embodiments, apolymeric material to be used in the present compositions and deliverysystems is an ethyl acetate and vinyl acetate solution such as“Duro-Tak” manufactured by Delasco Dermatologic Lab & Supply, Inc.

In certain embodiments, delivery systems of the invention comprisedelivery devices. In certain embodiments, the compositions of theinvention are delivered by an osmotic process at a controlled rate suchas by an osmotic pump. The system may be constructed by coating anosmotically active agent with a rate controlling semipermeable membrane.This membrane may contain an orifice of critical size through whichagent is delivered. The dosage form after coming into contact withaqueous fluids, imbibes water at a rate determined by the fluidpermeability of the membrane and osmotic pressure of the coreformulation. This osmotic imbibitions of water result in formation of asaturated solution of active material with in the core, which isdispensed at controlled rate from the delivery orifice in the membrane.

In certain embodiments, the compositions of the invention are deliveredusing biodegradable microparticles. In certain embodiment, the system toprepare microparticles consists of an organic phase comprised of avolatile solvent with dissolved polymer and the material to beencapsulated, emulsified in an aqueous phase. In certain embodiments,the biodegradable polymers that can be used for the microparticlematrix, comprises polylactic acid (PLA) or the copolymer of lactic andglycolic acid (PLAGA). The PLAGA polymer degrades hydrolytically overtime to its monomeric components, which are easily removed from the bodythrough natural life processes.

The preparation may also contain an absorption enhancer and otheroptional components. Examples of absorption enhancers include, but arenot limited to, are cyclodextrins, phospholipids, chitosan, DMSO, Tween,Brij, glycocholate, saponin, fusidate and energy based enhancingabsorption equipment.

Optional components present in the dosage forms include, but are notlimited to, diluents, binders, lubricants, surfactants, coloring agents,flavors, buffering agents, preservatives, stabilizing agents and thelike.

Diluents, also termed “fillers,” include, for example, dicalciumphosphate dihydrate, calcium sulfate, lactose, cellulose, kaolin,mannitol, sodium chloride, dry starch, hydrolyzed starches, silicondioxide, colloidal silica, titanium oxide, alumina, talc,microcrystalline cellulose, and powdered sugar. For administration inliquid form, the diluents include, for example, ethanol, sorbitol,glycerol, water and the like.

Binders are used to impart cohesive qualities to the formulation.Suitable binder materials include, but are not limited to, starch(including corn starch and pregelatinized starch), gelatin, sugars(including sucrose, glucose, dextrose, lactose and sorbitol),polyethylene glycol, waxes, natural and synthetic gums, e.g., acacia,tragacanth, sodium alginate, celluloses, and Veegum, and syntheticpolymers such as polymethacrylates and polyvinylpyrrolidone.

Lubricants are used to facilitate manufacture; examples of suitablelubricants include, for example, magnesium stearate, calcium stearate,stearic acid, glyceryl behenate, and polyethylene glycol.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents, with anionic surfactants preferred. Suitable anionicsurfactants include, but are not limited to, those containingcarboxylate, sulfonate and sulfate ions, associated with cations such assodium, potassium and ammonium ions. Particularly preferred surfactantsinclude, but are not limited to: long alkyl chain sulfonates and alkylaryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodiumsulfosuccinates, such as sodium bis-(2-ethylhexyl)-sulfosuccinate; andalkyl sulfates such as sodium lauryl sulfate.

Stabilizing agents such as antioxidants, include, but are not limitedto, propyl gallate, sodium ascorbate, citric acid, calciummetabisulphite, hydroquinone, and 7-hydroxycoumarin. If desired, thepresent compositions may also contain minor amounts of nontoxicauxiliary substances such as wetting or emulsifying agents,preservatives, and the like.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Analysis of Trehalose Dihydrate

Two (2) samples were brought in for analysis on Feb. 17, 2014.

The samples were kept at room temperature until analysis.

Sample Identification:

Analyst number Manufacturer Lot number Expiry date 14-01025 Hayashibara3G281 27 Jul. 2016 14-01026 Pfanstiehl 34943A August 2015

The samples were analyzed according to NF 31 page 2266-2267 duringAnalyst study 2014-003 “Trehalose NF Determination of Assay and RelatedSubstances by HPLC Method Verification”. Specificity was demonstratedfor glucose and maltotriose as required by the monograph. Thespecificity for trisaccharides produced by enzymatic modification ofstarch was not in the scope of the study. Sample 14-01026 was analyzedin six replicates and sample 14-01025 was analyzed in duplicate.

Results:

Sample 14-01025 Sample 14-01026 Hayashibara Pfanstiehl TestSpecifications lot 3G281 lot 34943A Assay 97.0-102.0% w/w on 98.2% 99.4%(conforms) anhydrous basis (conforms) Related Maltotriose ConformsConforms substances ≦0.5% w/w Any peak eluting before Conforms Conformstrehalose ≦0.5% w/w Glucose ≦0.5% w/w Conforms Conforms Any peak elutingafter Conforms Conforms trehalose ≦0.5% w/w

Example 2 Caballeta for IV Injection Certificate of Analysis

TABLE 2 Analysis of Trehalose Formulation Test Specification ResultAppearance of container Clear glass 30R vial with grey rubber Clearglass 30R vial with grey ATP007 stopper, aluminum seal and white fliprubber stopper, aluminum seal and off lid. white flip off lid.Appearance of contents Clear colourless liquid essentially free Clearcolourless liquid essentially ATP007 from visible particulate matterfree from visible particulate matter P537 Identity Retention time of theP537 peak ±5% Retention time of the P537 ATP1323 of standard peak iswithin ±5% of the P537 standard peak P537 Content Assay 90.0% to 110.0%label claim 99.8% ATP1323 P537 Related Substances Report individualimpurities Maltotriose: None detected (% label claim) ≧0.05% label claimUnknown RRT0.90: 0.1% ATP1323 Report total impurities Glucose: Nonedetected Maltotriose and other polysaccharides Total Impurities: 0.1%eluting before P537: ≦0.5% Glucose and peaks eluting after P537 ≦0.5%Total impurities s 2.0% pH 4.5 to 7.0 6.2 ATP164 Osmolality 280-330mOsm/kg 289 mOsm/kg ATP841 Particulate matter Particulates ≧10 μm: NMT6000 ≧10 μm: 7 USP <788>, Ph. Eur Particulates ≧25 μm: NMT 600 ≧25 μm: 02.9.19 Extractable Volume Not less than 30 ml 32 ml USP <1>, Ph. Eur2.9.17 Endotoxins <0.24 EU/ml Point 1 tray 1: <0.1 EU/ml USP <85>, Ph.Eur 2.6.14 Point 4 tray 31: <0.1 EU/ml Point 8 tray 72: <0.1 EU/mlSterility Complies No growth USP <71>, Ph. Eur 2.6.1

Example 3 Preclincal Pk Study

The plasma and muscle concentrations of trehalose in male Sprague-Dawley(SD) rats was determined after intravenous bolus (IV) and oral gavage(PO) administration.

All applicable portions of the study confirmed to the followingregulations and guidelines regarding animal care and welfare: AAALACInternational and NIH guidelines as reported in the “Guide for the Careand Use of Laboratory Animals,” National Research Council ILAR, Revised1996.

The study included 42 SD rats (male, 250 to 350 grams in weight, theShanghai SLAC Laboratory Animal Co. Ltd.). Animals were administeredwith a volume of 5 ml/kg trehalose formulation (trehalose dihydrate insterilized water at 200 mg/mL) to achieve a nominal dose of 1 gr/kg,intravenously or orally.

Blood samples were collected after each dose administration andprocessed for plasma. Muscle samples (hind leg muscle) were collectedand homogenized. The concentrations of trehalose in plasma and musclehomogenate samples were analyzed by qualified bioanalytical LC/MS/MSmethods.

Pharmacokinetics Data Analysis

Plasma concentration data of trehalose was subjected to a noncompartmental pharmacokinetic analysis using WinNonlin software program(version 6.3, Pharsight, Mountain View, Calif.). Zero-time interceptconcentration (C0), volume of distribution (Vdss), Clearance (Cl), peakplasma concentrations (Cmax) and the corresponding peak times (Tmax),terminal half-life (T½), mean residence time (MRT) from time zero to thelast time point (MRT0-last), MRT from time zero to infinity (MRT0-inf),the area under the plasma concentration-time curve (AUC) from time zeroto the last time point (AUC0-last) and AUC from time zero extrapolatedto infinity (AUC0-inf) were calculated using the linear/log trapezoidalrule. Nominal sampling times were used to calculate all pharmacokineticparameters since there was not any deviation larger than 5% between theactual and nominal sampling times.

The values of muscle to plasma concentration and AUC ratio (M/P ratio)were both calculated.

Trehalose Concentration in Plasma and Muscle

Pharmacokinetic parameters of trehalose in the plasma and musclefollowing single intravenous or oral administration of trehalosedihydrate solution (200 mg trehalose dihydrate per 1 mL sterilizedwater) at 1000 mg/kg to male SD rats are presented in Table 3 below.

Individual and mean plasma concentrations of trehalose followingintravenous or oral administration of trehalose dihydrate solution (200mg trehalose dihydrate per 1 mL sterilized water) at 1000 mg/kg to maleSD rats are presented in Table 3 and shown graphically in FIG. 1.

Individual and mean muscle concentrations of trehalose following singleintravenous or oral administration of trehalose dihydrate solution (200mg trehalose dihydrate per 1 mL sterilized water) at 1000 mg/kg to maleSD rats are presented in Table 3 as well. Plasma and muscleconcentrations comparison for trehalose following single intravenous ororal administration of trehalose dihydrate at 1000 mg/kg to male SD ratsare and shown graphically in FIGS. 2 to 3.

Following a single intravenous dose of trehalose solution (200 mgtrehalose dihydrate per 1 mL sterilized water) at 1000 mg/kg to malefasted SD rats in tested groups 1 to 5, trehalose showed a totalclearance (CI) of 17.2 mL/min/kg (approximately 31.3% of rat liver bloodflow (=55 mL/min/kg)), with the averaged elimination half life (T_(1/2))of 2.07 hours. The C₀ was 1,370,000 ng/mL.

The volume distribution (V_(dss)) was at 0.685 L/kg. The mean plasmaexposure AUC_(0-last) (48 hr) was 778,000 ng·hr/mL.

With an oral administration of trehalose dihydrate solution (200 mgtrehalose dihydrate per 1 mL sterilized water) to male SD rats in testedgroups 6 to 10, trehalose maximum plasma concentration (C_(max)=4,280ng/mL) was attained at 0.5 hour post dose (T_(max)). The AUC_(0-last)(3hr) was 4,520 ng/mL·hr. The absolute bioavailability of trehalose wasestimated to be as low as 0.601%.

The pharmacokinetic properties of trehalose demonstrated a rapidabsorption with a time to reach peak plasma concentrations, but theabsolute oral bioavailability was very low, which noted that thecompound may undergo a significant presystemic metabolism.

Following a single intravenous dose of trehalose dihydrate solution (200mg trehalose dihydrate per 1 mL sterilized water) at 1000 mg/kg to maleSD rats in tested groups 1 to 5, the C_(max) of trehalose in muscle was3730 ng/mL, which was observed at 8 hours (T_(max)) post dose. Themuscle exposure AUC_(0-last) (48 hr) was 107,000 ng·hr/mL with theelimination half life 33.8 hours. The PK parameters of muscle samples indrug treated oral group (Groups 6 to 10) could not be calculated becausethey were below LLOQ

The mean ratios of muscle trehalose concentration to plasmaconcentration ranged from 2.88 to 3.76 in male SD rats followingintravenous administration. Muscle to plasma concentration ratios fortrehalose upon oral administration were below LLOQ and not calculable.

Conclusions

Following intravenous or oral administrations of trehalose dihydratesolution in sterilized water at 1000 mg/kg to male SD rats, trehalose inplasma and muscle tissue were determined Plasma glucose was alsomonitored for each sample from study animals of drug treated groups. Thefollowing conclusions can be made:

First, following IV administration, the total clearance (Cl) oftrehalose was 17.2 mL/min/kg, accounting for approximately 31.3% ofliver blood flow, a moderate value of hepatic extraction ratio. TheV_(dss) and T_(1/2) were 0.685 L/kg and 2.07 hours respectively. Themean plasma exposure AUC_(0-last) was 778,000 ng·hr/mL.

Following oral administration, trehalose demonstrated a rapid absorptionwith T_(max) observed at 0.50 hours post dose, but the absolute oralbioavailability was as low as 0.601%, suggesting presystemic metabolismmay play an important role. T_(1/2) of trehalose was markedly shortenedin oral administration rats in comparison to the intravenous group.

Following IV administration, the mean ratios of muscle trehaloseconcentration to plasma concentration ranged from 2.88 to 3.76 in maleSD rats.

Finally, it was observed that trehalose dihydrate was well tolerated bythe rats at the given dosage.

TABLE 3 Pharmacokinetic parameters Matrix Plasma Muscle Group ID IV POIV PO C₀ (ng/mL) 1370000 — — ND C_(max) (ng/mL or ng/g) — 4280 3730 NDT_(max) (h) — 0.500 8.00 ND T_(1/2) (h) 2.07 0.740 33.8 ND CI(mL/min/kg) 17.2 — — ND V_(dss) (L/kg) 0.685 — — ND AUC_(0-last) (ng ·h/mL or 778000 4520 107000 ND ng · h/g) AUC_(0-inf) (ng · h/mL or 7810004870 183000 ND ng · h/g) MRT_(0-last) (h) 0.618 1.04 21.1 ND MRT_(0-inf)(h) 0.666 1.26 52.6 ND AUC_(0-inf)/AUC_(0-last) 100 108 171 ND (%)^(c)Bioavailability (%) — 0.601 — — ^(d)AUC ratio — — 0.234 NDAbbreviations: ND = Not determined; ^(c)Bioavailability (%) wascalculated with mean AUC_(0-inf) and nominal dose; ^(d)AUC Ratio =Muscle AUC_(0-inf)/Plasma AUC_(0-inf); AUC_((0-inf)) >120% ofAUC_((0-last)).

Thus, as demonstrated in Table 3, the trehalose in the IV administeredformulation showed T_(1/2) of 2.07 hour in plasmas, over two-fold higherthan the plasma T_(1/2) obtained for trehalose in the orallyadministered formulation (0.740 hours). In addition, the muscle T_(1/2)obtained for the trehalose in the IV administered formulation was 33.8hours. The AUC values obtained for plasma and muscle when theformulation was administered IV were also significantly higher than therespective AUC values of formulation administered orally.

In addition, as demonstrated in FIG. 1 the mean plasma concentration ofthe trehalose in the IV administered formulation is higher than the meanplasma concentration of the trehalose in the orally administeredformulation at each of the tested time points.

Interestingly, FIG. 2, which demonstrates plasma versus muscleconcentrations of trehalose for a trehalose formulation administeredintravenously, shows that muscle concentrations are higher than plasmaconcentrations of trehalose. Plasma and muscle concentrations oftrehalose were undetectable for a trehalose formulation administeredorally.

Summary of Pre-Clinical Study

Study Details Administered compound Trehalose Sponsor BBP Sponsor StudyNo. NA Wuxi DMPK Study No. BBP-20140220-RPK Species Male SD Rat, fastedStudy Group IV PO Nominal dose (mg/kg) 1000 1000 Administered dose(mg/kg  804  778 Formulation IV 200 mg/mL in water for injection, clearsolution Formulation PO 200 mg/mL in water for injection, clear solutionIn-life start date Mar. 11, 2014 Bioanalytical Details Analyte compoundTrehalose Batch No. 34943A Molecular weight 342.3 Formular weight 378.33Salt factor  1.11 Purity  90.5 Analytical technique LC-MS/MS MatrixPlasma (EDTA-K₂) PK Calculation Settings Program Phoenix WinNonlin 6.3Model IV-Noncompartmental model 201 (intravascular input) PO-Noncompartmental model 200 (extravascular input) Calculation Linear/logtrapezoidal method Dose used (WNL Nominal dosage “BQL” was excluded inthe PK parameters and mean plasma concentration calculation. Comments 1.The value of all the samples “predose” was below the lower limit ofquantitation (LLOQ = 100 ng/mL) 2. Stock solution for standard curve wasdissolved in DMSO with active content considered at 81.9%. However, ourprotocol calculated active content at 90.5%. Finally, all the measuredvalue was adjusted with following, Actual concentration value = Measuredvalue/0.905

Example 4 Trehalose Disposition

Oral absorption: When ingested orally, most of the sugar is notassimilated as a disaccharide into the blood stream. Rather, it isenzymatically hydrolyzed in the small intestine by a trehalose-specificdisaccharidase (trehalase) into two d-glucose molecules, which aresubsequently absorbed and metabolized. Trehalase is found in mostanimals at the brush border of the intestinal mucosa, as well as in thekidney, liver and plasma. Although trehalose does not occur in mammaliancells, humans have the enzyme trehalose in intestinal villae cells andin kidney brush border cells, probably to handle ingested trehalose[11,14,15].

Biotransformation and excretion: When trehalose enters blood circulationit is rapidly converted to glucose by trehalase in serum, kidney, liverand bile, depending on the species. In animals lacking trehalaseactivity in the kidney such as rats, most of the intravenous trehaloseis excreted in the urine, proportional to plasma concentration. Inanimals where renal trehalase activity exists (such as guinea-pigs andrabbits), only a very small fraction of trehalose is recovered in theurine. When rabbits were given 500 mg of trehalose intravenously(corresponding to 200±300 mg/kg body weight) the compound was clearedfrom the plasma within 60 min, and none was detected in the urine [15].

Example 5 Animal Safety

The safety and tolerability of trehalose has been extensivelyinvestigated. A detailed review of safety in animals and humans ispresented in the Cabaletta Investigator's brochure, incorporated hereinby reference (the term Cabaletta as used herein signifies a 10% IVsolution of trehalose).

Animal toxicity: Trehalose LD50 was examined in mice, rats and dogs.Neither species showed any signs of toxicity and no deaths occurredafter oral and intravenous administration. The results are summarized inTable 1:

TABLE 1 LD50 of trehalose in animals SPECIES ROUTE LD50 (mg/kg bw) MouseOral >5000 Mouse Intravenous >1000 Rat Oral >16000 Rat Oral >5000 RatIntravenous >1000 Dog Oral >5000 Dog Intravenous >1000

Human Safety and Use:

Trehalose is recognized as a safe food ingredient as well as a GRASmaterial used in the pharmaceutical industry as an excipient for oral,intraocular and I.V. drug formulations.

In several studies, healthy volunteers were given oral doses oftrehalose ranging from 10 to 60 gr. Apart from mild abdominal symptoms(e.g. flatulence, distension, borborygmus and occasional diarrhea) noother safety issues were reported.

Trehalose has been used as a protein stabilizer in several commerciallyavailable protein drugs for over a decade and its safety has repeatedlybeen established in patient populations at advanced stages of malignantdiseases, hemophilia and related clotting disorders. These drugs areapproved for use for several years, and are sometimes given to patientsas frequently as every 8 hours through 2-3 weeks intervals.

Example 6 Clinical Study

A three-center, multi-national, randomized, double-blind, doseescalation and parallel-group dose-controlled study will be conducted toassess the safety, tolerability, and efficacy of IV Cabaletta® inPatients with Oculopharyngeal Muscular Dystrophy (OPMD). The study willbe comprised of an Exploratory phase (screening period and a treatmentperiod), an interim analysis, and a Pivotal phase (second treatmentperiod and a follow-up period).

Up to 30 adult patients with OPMD will be enrolled into the study ateach of the three study sites. A minimum of 42 patients will be enrolledin total.

Inclusion Criteria

1. Males and females

2. 18-80 years (inclusive) of age

3. Genetically diagnosed with OPMD

4. Moderate dysphagia (abnormal drinking test at screening and on thefirst dosing day, before drug administration)

5. Patients must be ambulatory, and capable of performing the musclefunctional and strength assessments

6. Patients who provide written informed consent to participate in thestudy

7. Body Mass Index (BMI)<30 kg/m2

8. Female patients of child-bearing potential must have a negative serumpregnancy test at screening

9. Male and females must agree to use acceptable birth control

10. Patients must be able to understand the requirements of the studyand be willing to comply with the requirements of the study

Exclusion Criteria

1. Diabetes mellitus Type 1 or 2

2. Other major diseases, e.g. renal failure (creatinine clearance <60ml/min), liver failure and chronic liver diseases (e.g. hepatitis B orC), HIV carriers, tuberculosis, SLE, rheumatoid polyarthritis,sarcoidosis, collagenosis

3. Uncontrolled heart disease, e.g., CHF

4. Other neuromuscular diseases

5. Other disorders associated with esophageal dysphagia: e.g.gastroesophageal reflux (GERD), esophageal stricture due to mechanicalor chemical trauma, infection (e.g. esophageal moniliasis), drug-induceddysphagia (e.g. bisphosphonates), esophageal rings and webs, spasticmotility disorders of the esophagus.6. History of malignancy7. History of neck irradiation8. Pregnant or currently lactating women9. Obesity (BMI≧30) and associated morbidity10. Prior pharyngeal myotomy11. Weight loss of more than 10% in the last 12 months.12. Known hypersensitivity to any ingredients in the injection

Dose

Cabaletta®, a 10% IV solution of trehalose, will be administered once aweek for 72 weeks. Study drug will be delivered over approximately 80minutes. Doses used in the study:

Dose Study week Initial dose (unblinded)  8 g Week 1 Second dose(unblinded) 15 g Week 2 treatment ( 30 g Week 3 to week 24 Randomized 30g or no treatment Week 25-72, discontinuation

Study Objectives

Exploratory Phase (24 Weeks) Study Objectives

Primary

1. To determine the safety and tolerability of Cabaletta in OPMDpatients after a single (8 gr) IV administration.

2. To determine the safety and tolerability of Cabaletta in OPMDpatients after a single (15 gr) IV administration

3. To determine the safety and tolerability and 30 g IV Cabaletta inOPMD patients after repeated weekly dosing.

4. 5. To obtain data on the pharmacokinetics of trehalose.

6. To determine the pharmacokinetics of trehalose (Israel site only)

Secondary

1. To determine the effect of Cabaletta on the progression of OPMD,assessed by measuring dysphagia, swallowing-related quality of life(SWAL-QOL), and muscle function and strength.

2. At the end of the exploratory phase patients will be randomized into2 groups: 1 group will continue with the weekly injections of 30 gramsand the other will not get treatment however they will monitored throughthe pivotal phase in the same way the treated patients will be. Pivotalphase (48 weeks) Study Objectives

Primary

1. To determine the effect of Cabaletta on the progression of OPMD,assessed by measuring dysphagia, swallowing-related quality of life(SWAL-QOL), and muscle function and strength

2. To compare the efficacy 30 g Cabaletta with no treatment.

Secondary

1. To determine the safety and tolerability of 30 g IV Cabaletta in OPMDpatients after repeated weekly dosing.

2. The study will be comprised of an Exploratory phase (screening periodand a treatment period), an interim analysis, blinded randomization anda Pivotal phase (second treatment period and a follow-up period) asfollows:

Study Procedures

Exploratory Phase

Screening Period (Week-4/Day-28 to Week 0/Day 0)

Screening assessments will be conducted over two visits within 28 daysprior to the start of therapy, as specified in the Schedule ofAssessments.

Treatment Period 1 (Week 1 to Week 24)

All eligible patients will receive study treatment once a week.

Initially, all eligible patients will receive one dose of Cabaletta 8 gover one week, followed by 15 g Cabaletta over the next week (Visits 3and 4). If no safety concerns arise, at Visit 5/Week 3 all patients willreceive to receive Cabaletta 30 g for 24 weeks. The first 4 infusionswill be done at the clinic under the direction of the studyinvestigator. The patients must return to the clinic once a month fordrug infusion and study assessments, as indicated in the Schedule ofProcedures; all other weekly infusions may be done in the patient's homeor in the clinic.

Interim Analysis

The interim analysis will be conducted when the first patient enrolledinto the study has completed 6 months of therapy, and the last subjectenrolled has completed at least 3 months of therapy. Both safety andefficacy will be examined in the interim analysis.

Pivotal Phase

Treatment Period 2 (Week 25 to Week 72)

Patients Will be Randomized into Two Arms: Continued Treatment Arm andDiscontinuation Arm (No Treatment Control)

Patients assigned to the continued treatment arm will continue weekly IVinfusions of Cabaletta at home or at the hospital, except for Visits 32,40, 48, 60 and 72. Study procedures will be done as specified in theSchedule of Assessments.

Patients assigned to the no-treatment control will not be gettingadditional infusion however they will be followed up and monitored insame schedule planned for the treatment arm,

Follow-Up Period (4 Weeks Post-Dose)

Patients will be seen at a post-treatment follow-up visit (Visit 75), 4weeks after the final dose.

Safety and Tolerability Outcome and Assessments

The primary safety endpoint is the frequency, severity, and duration ofadverse events (AEs), including clinically significant laboratoryabnormalities after administration of Cabaletta.

Safety will be evaluated on the basis of the following assessments:

AEs and concomitant medications: Continuous (starting from informedconsent signature until end of study)

Physical examination: on screening (Visit 1), on the first 4 dosingvisits, and then once monthly in the experimental phase, at the interimanalysis and once every 6-12 weeks at the pivotal phase as outlined inthe schedule of assessment in appendix A.) and End of Study (EOS,Visit75).

12-lead ECG: at screening (Visit 1), during the first dosing with 15 gand 30 g (Visits 4 and 5), at the interim analysis (Visit 26), and atthe end of study evaluation (Visit 75).

Vital signs: on screening (Visit 1) and on each visit until end ofstudy. On the first 4 dosing visits, vital signs will be assessed priorto the Cabaletta administration, every 30 min during administration and30 min following the drug administration.

Safety laboratory evaluations will be conducted according to theSchedule of Procedures. Evaluations will include: complete blood count(CBC) with differential, electrolytes (Na, K, Cl), BUN, creatinine,glucose, liver function tests (ALT, AST, total bilirubin, directbilirubin, alkaline phosphatase, and serum albumin), and dipstickurinalysis.

Pre- and post-dose blood glucose will be measured at the first 3 dosingvisits; blood glucose will also be assessed at the End of Studyevaluation (Visit 75). Urine pregnancy will be done at Screening (Visit1), baseline (Visit 3), and EOS (Visit 75).

Safety data will be reviewed periodically by an independent data safetymonitoring board.

Evaluation

The following assessments will be performed:

Penetration Aspiration Score (Using Videofluoroscopy)

SWAL-QOL

Muscle Timed Functional and Strength Assessments

Changes compared to baseline will be measured for each patient, and thetotal change in scores for the treatment groups in each pre-determinedefficacy endpoint will be statistically analyzed.

The following additional assessments, thought to be supportive innature, will be performed at the times specified in the Schedule ofAssessments:

-   -   Weight    -   Drinking test    -   Percutaneous Core Needle Biopsy (PCNB) will be performed to        obtain muscle fiber for histology.

Pharmacokinetics

The pharmacokinetic of trehalose will be assessed in patients (Israelisite only) at the randomized dose of 15 g or 30 g Cabaletta. Trehaloseblood concentration will be measured pre-dose (up to 60 minutes beforestudy drug administration); and every 30 minutes after dosing isinitiated, for 5 hours or until glucose levels return to normal,whichever occurs first.

Statistical Methods

All measured variables and derived parameters will be listedindividually and, if appropriate, tabulated by descriptive statistics.For descriptive statistics summary tables will be provided giving samplesize, absolute and relative frequency and 95% Confidence Interval (CI)for categorical variables and sample size, arithmetic mean, standarddeviation, coefficient of variation (if appropriate), median, minimumand maximum, percentiles and 95% CI for means of continuous variables.

Rate of subjects with any adverse event and potentially clinicallysignificant laboratory results with 95% CI will be calculated bytreatment period.

Exploratory statistical analyses of data may be undertaken asappropriate.

All tests applied will be two-tailed, and p value of 5% or less will beconsidered statistically significant.

Schedule of Assessments

Assessment Screen- ing and Base- line Exploratory Phase Visit No. 1 2 34 5 6 7 8 9 10 11-13 14 15-17 18 19-21 22 Dosing Week −4 1 2 3 4 5 6 7 89-11 12 13-15 16 17-19 20 Informed Consent X — — — — — — — — — — — — — —— Demographics X — — — — — — — — — — — — — — — Medical history X — — — —— — — — — — — — — — — Weight X — X — — X — — — X — X — X — X Vital signsX X X X X X X X X X X X X X X X Physical X — X X X X — — — X — X — X — Xexamination 12-Lead ECG X — — X X — — — — — — — — — — — Safety labs X —X X X X — — — — — X — — — — Urine pregnancy X — X — — — — — — — — — — —— — Blood glucose — X X X — — — — — — — — — — — HIV, HBsAg, X — — — — —— — — — — — — — — — HCVAb Drinking test X — X — — X — — — X — X — X — XInclusion/Exclusion X — X — — — — — — — — — — — — — Videofluoroscopy — X— — — — — — — — — — — — — — SWAL-QOL — X — — — — — — — — — — — — — —Muscle — X — — — — — — — — — — — — — — function/strength Muscle Biopsy —X — — — — — — — — — — — — — — Randomization — — — — X — — — — — — — — —— — Dosing at study site — — X X X X X — X — X — X Dosing at patient's —— — — — — X X X — X — X — X — home or study site Adverse Events — X X XX X X X X X X X X X X X Concomitant X X X X X X X X X X X X X X X XMedications Pharmacokinetics — — — — — — X — — — — — — — — — (Israelsite only) Assessment Explo- End ratory of Phase Pivotal Phase StudyVisit No. 23-25 26 27-33 34 35-41 42 43-61 62 63-73 74 75 Dosing Week21-23 24 25-31 32 33-39 40 41-59 60 61-71 72 — Informed Consent — —Interim — — — — — — — — — Demographics — — Analysis and — — — — — — — —— Medical history — — randomuzaion — — — — — — — — — Weight — X — X — X— X — X X Vital signs X X X X X X X X X X X Physical — X — X — X — X — XX examination 12-Lead ECG — X — — — — — — — — X Safety labs — — — X — X— X — X X Urine pregnancy — — — — — — — — — — X Blood glucose — — — — —— — — — — X HIV, HBsAg, — — — — — — — — — — — HCVAb Drinking test — X —X — X — X — X X Inclusion/Exclusion — — — — — — — — — — —Videofluoroscopy — X — — — — — — — — X SWAL-QOL — X — — — — — — — — XMuscle — X — — — — — — — — X function/strength Muscle Biopsy — X — — — —— — — — X Randomization — — — — — — — — — — — Dosing at study site — X —X — X — X — X — Dosing at patient's X — X — X — X — X — — home or studysite Adverse Events X X X X X X X X X X X Concomitant X X X X X X X X XX X Medications Pharmacokinetics — — — — — — — — — — — (Israel siteonly)

Example 7 Determination of Endotoxin Level

It is accepted that the maximal allowed level of endotoxin informulations administered intravenously is 5 endotoxin units (EU) per kgbody mass per hour (5 EU/kg/hr). In order to determine the theoreticalmaximum endotoxin level IV per kg body mass/hour (K) in trehaloseformulation (solution of trehalose dihydrate in sterilized water), thefollowing calculations were made:

TABLE 4 Calculation of maximal endotoxin levels in trehaloseformulations 30 gr trehalose Endotoxin contribution 15 gr trehaloseformulation formulation 2.4 EU/gr (trehalose) 36 72 0.5 EU/ml (solvent)75 (in 150 ml) 150 (in 300 ml) Total EU in formulation 111 222 Assuming75 min infusion 88.8 EU/hr 177.6 EU/hr K for 60 kg body weight 1.5 3.0 Kfor 50 kg body weight 1.8 3.6 K for 40 kg body weight 2.2 4.4

As indicated in Table 4 above, endotoxin level per ml in trehaloseformulations prepared with standard solvents (e.g. water, saline, etc.)is 0.74 EU/ml. Assuming a moderate infusion rate of 75 minutes, for abody weight of 60, 50 and 40 kg the endotoxin level in trehalose 10%(w/v) formulations is 1.5, 1.8 and 2.2 EU/kg/hr, respectively, for aformulation comprising 15 gr trehalose and 3.0, 3.6 and 4.4 EU/kg/hr,respectively, for a formulation comprising 30 gr trehalose.

Accordingly, under the maximum rate planned, the endotoxin level for abody weight of 60, 50 and 40 kg will be 2, 2.4 and 3 EU/kg/hr,respectively, for a formulation comprising 15 gr trehalose (in 150 mlsolvent) and 4, 4.8 and 6 EU/kg/hr, respectively, for a formulationcomprising 30 gr trehalose (in 300 ml solvent).

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method of alleviating a sign or symptom ofoculopharyngeal muscular dystrophy (OPMD) by intravenously administeringto a subject in need thereof an aqueous formulation for intravenousinjection comprising a single active ingredient consisting of trehalose,wherein the formulation (i) has a pH about 4.5 to 7.0, (ii) containsless than 0.75 endotoxin units per mL, and (iii) is administered at aper administration dose of between 5 to 50 grams trehalose, wherein theadministration is completed within less than 120 minutes.
 2. The methodof claim 1, wherein the formulation is administered once weekly.
 3. Themethod of claim 2, where the formulation is administered at a peradministration dose of about 0.5 gram trehalose per kilogram bodyweight.
 4. The method of claim 1, wherein the per day dose is 8, 15 or30 grams.
 5. The method of claim 1, wherein the rate of administrationis such that the maximum endotoxin level is less than 5 EU per kilogramof body weight per hour.
 6. The method of claim 1, wherein the trehaloseis at 10% (w/v).
 7. The method of claim 1, wherein the formulation hasan osmolality of about 280-330 mOsm/kg.